Most agricultural and horticultural crops are under a constant threat due to fungal attack. To protect the crops from significant losses due to fungal disease, the crops and sometimes the soil in which the crops are grown are periodically treated with large amounts of fungicides. These fungicides form a heavy burden on costs of crop growing, and more importantly on the environment and the growers. Moreover the treatment is very labor intensive. Therefore, there is a need for less costly and safer methods to protect plants from fungal attack which, preferably, are devoid of the need of repeated human involvement.
Induced Resistance
In plants generally several types of resistance against pathogens occur; non-host resistance, "horizontal" or partial resistance and "vertical" resistance. None of these forms of resistance is particularly well understood in molecular terms. In addition to these constitutively expressed forms of resistance there is a resistance mechanism that can be induced by certain pathogenic infections as well as by a number of biotic and abiotic factors. This induced resistance is very broad and is directed against various pathogens, including fungi. This is further illustrated below. Inoculation of the lower leaves of a hypersensitively reacting tobacco cultivar (Nicotiana tabacum cv Samsun NN) with tobacco mosaic virus (TMV) results in the formation of local lesions on the inoculated leaves. The non-inoculated leaves appear resistant to a second infection with TMV after 3 days; this resistance lasts at least twenty days, and an optimal resistance is obtained after 7 days. The resistance against the second infection is also directed to other viruses, such as tobacco necrosis virus, tobacco ringspot virus (Ross & Bozarth, 1960; Ross, 1961), and fungi, such as Thielaviopsis basicola (Hecht & Bateman, 1964), Phytophthora nicotianae and Peronospora tabacina (McIntyre & Dodds, 1979; McIntyre et al., 1981).
The phenomenon of induced resistance has been studied in numerous other host plants and in combination with several other pathogens as well (Kuc, 1982; Sequeira, 1983). The general picture emerging from these studies is that a hypersensitive response is accompanied by resistance against a broad range of pathogens, irrespective of the type of pathogen having caused the first infection.
Proteins Expressed Concomitant with Induced Resistance
Together with the resistance a great number of proteins is synthesized, which are absent before infection.
Roughly three categories of proteins can be discerned.
1) Key-enzymes in the synthesis of secondary metabolites, such as phytoalexins, which exhibit an antimicrobial effect, and precursors of lignin, which is used in the reinforcement of plant cell walls after pathogen invasion. These enzymes, or their messenger RNAs are mainly found in cells in the immediate vicinity of the site of infection (Elliston et al., 1976; Cramer et al., 1985; Bell et al., 1986). PA1 2) Hydroxyproline rich glycoproteins (HRGPs) or extensins, which can be incorporated into the cell wall and possibly function there as a matrix for the attachment of aromatic compounds like lignin (Fry, 1986). HRGPs are important structural components of plant cell walls, and their accumulation occurs in reaction to fungi, bacteria and viruses (Mazau & Esquerre-Tugaye, 1986). In contrast to the situation with the key-enzymes mentioned above, HRGPs and their mRNAs are found in substantial amounts in non-infected parts of the plant as well as around the site of infection (Showalter et al., 1985). PA1 3) A third group of induced genes encodes proteins which accumulate both inside the cells and in the apoplastic space. Among these proteins are hydrolytic enzymes such as chitinases and glucanases. After a necrotic infection these enzymes can often be found throughout the plant, including the non-infected parts, in higher concentrations than before infection. Increased synthesis of these enzymes appears to be induced also by microbial elicitors, usually fungal cell wall preparations (Darvill & Albersheim, 1984; Toppan & Esquerre-Tugaye, 1984; Mauch et al., 1984; Chappel et al., 1984; Kombrink & Hahlbrock, 1986; Hedrick et al., 1988).
Structure of Fungal Cell Walls
The cell walls of fungi are known to consist of a number of different carbohydrate polymers. Most fungi, with the exception of the Oomycetes, contain considerable amounts of chitin. Chitin is a polymer of N-acetyl glucosamine molecules which are coupled via .beta.-1,4 linkages and, in fungal cell walls, are often associated with .beta.-1,3/.beta.-1,6 glucan, polymers of glucose with .beta.-1,3 and .beta.-1,6 linkages. Fungi from the group of Zygomycetes do not contain glucans with .beta.-1,3 and .beta.-1,6 linkages, while in most of the oomycetes the glucans are associated with cellulose (for an overview, vide: Wessels and Sietsma, 1981).
In Vitro Degradation of Isolated Fungal Cell Walls
It has been known for a long time that isolated cell walls of fungi can be degraded in vitro by plant extracts (Hilborn & Farr, 1959; Wargo, 1975; Young & Pegg, 1982) and also by chitinase and .beta.-1,3-glucanase preparations from microbial origin (Skujins et al., 1965; Hunsley & Burnett, 1970; Jones et al., 1974).
More recently a purified endo-.beta.-1,3-glucanase from tomato in combination with an exo-.beta.-1,3-glucanase of fungal origin were shown to be capable of hydrolysing isolated cell walls of the fungus Verticillium albo-atrum. Each of the preparations separately did not have activity (Young & Pegg, 1982). A purified .beta.-1,3-glucanase from soybean (Keen & Yoshikawa, 1983), as well as a purified chitinase from bean (Boller et al., 1983) have also been shown to be capable of degrading isolated cell walls of fungi in vitro. When pea chitinase and .beta.-1,3-glucanase were tested on isolated cell walls of Fusarium solani, both appeared to be active; in combination they appeared to work synergistically (Mauch et al., 1988b).
It is not known whether these hydrolytic enzymes can degrade the polymer compounds in cell walls of living fungi effectively, if at all.
Inhibition of Fungal Growth on Synthetic Media by Chitinases and Glucanases from Plant Origin
Some chitinases and glucanases of plant origin are capable of inhibiting the growth of fungi on synthetic media. Chitinase purified from bean is capable of inhibiting the growth of the fungus Trichoderma viride in agar plate assays (Schlumbaum et al., 1986). A combination of chitinase and .beta.-1,3-glucanase, both purified from pea pods, do inhibit the growth of some fungi on agar plates, whereas other fungi are not inhibited. The Ascomycete Cladosporium cucumerinum appeared slightly sensitive, while the oomycetes Phythophthora cactorum, Pythium aphanidermatum and Pythium ultimum are insensitive. Pea chitinase alone-has effect on the growth of T.viride, while .beta.-1,3-glucanase inhibits the growth of Fusarium f.sp. pisi. It was established that in these assays the inhibition of fungal growth was due to lysis of the hyphal tips (Mauch et al., 1988b). Apparently the hydrolytic enzymes do have access to their substrate in the cell walls of living fungi, when grown on synthetic media, although at least some of the active plant hydrolytic enzymes seem to be specific to certain fungi.
Little is known about the effect of hydrolytic enzymes on fungi in the biotrope, i.e. in the soil or on plant leaves, and although some of these enzymes are putative candidates for a role in fungal resistance, evidently, not all chitinases and glucanases have activity against living fungi.
Possibly, the stage and site of infection at which hydrolytic enzymes come into contact with the invading fungus may be of great importance.
Occurrence of Chitinases and Glucanases in Plants
As far as known, chitinases and .beta.-1,3-glucanases occur in most if not all plant species; both in monocotyledonous and dicotyledonous plants. At least two classes of chitinases and two classes of glucanases can be discriminated: intracellular and extracellular. Both chitinase and glucanase genes of one. particular class appears to be encoded by gene families.
Natural Expression of Chitinases Genes and Glucanase Genes in Plants
Chitinase and glucanase genes are known to be expressed in plants both constitutively and in a strictly regulated fashion.
Chitinases and .beta.-1,3-glucanases are constitutively synthesised in roots of tobacco plants (Felix and Meins, 1986, Shinshi et al., 1987,; Memelink et al., 1987, 1989). Nevertheless tobacco plants are not resistant to infection of Phytophthora parasitica var. nicotianae (a root pathogen of tobacco). However, resistance against this pathogen can be induced in tobacco plants, following inoculation with TMV (McIntyre & Dodds, 1979). This suggests that a complex of yet unknown factors other than, or in addition to, chitinases and glucanases, may be involved in fungal resistance.
On the other hand, plant species are known which seem to be resistant to fungal infection, although no significant increase in the levels of chitinases or glucanases can be observed. For instance, in tomato a compatible interaction with the fungus Phytophthora infestans causes a systemic resistance (Christ & Mosinger, 1989), i.e. a resistance to infection throughout the whole plant, although chitinases or glucanases cannot be detected in such leaves (Fischer et al., 1989). Apparently there is no clear correlation between expression of the genes encoding hydrolytic enzymes and fungal resistance.
In addition to these observations, some chitinases exhibit a regulated expression pattern which does not immediately suggest a correlation with fungal resistance.
For example, genes encoding chitinases are known to be expressed in a developmentally regulated manner in, inter alia, tobacco flowers (Lotan et al., 1989). Glucanases are known to occur in large quantities in seedlings of barley (Swegle et al., 1989; Woodward & Fincher, 1982; Hoj et al., 1988, 1989).
In tobacco cell suspensions the synthesis of intracellular chitinases and glucanases can be inhibited by the addition of cytokinins or auxins (Mohnen et al., 1985; Felix & Meins, 1986; Shinshi et al., 1987; Bauw et al, 1987).
The synthesis of the same hydrolytic enzymes can be induced by cytokinin when this hormone is added to the growth medium in which normal tobacco plants are grown axenically. Under certain circumstances the plant hormone ethylene can also induce the synthesis of chitinase and glucanase (Felix & Meins, 1987).
In the roots and lower leaves of both soil-grown and axenically grown tobacco plants, intracellular chitinases and glucanases can be detected, while in upper leaves they can not be detected at all, or to a much lesser extent (Felix & Meins, 1986; Shinshi et al 1987; Memelink 1987, 1989). Thus, there is also organ-specific expression of the intracellular chitinases and glucanases.
The regulation of expression of the genes coding for extracellular chitinases and glucanases is hardly, or not at all, influenced by cytokinin (Memelink et al 1987., 1989). In tobacco flowers the extracellular chitinases are expressed specifically in anthers, sepals and the ovary.
Thus, there is at least an organ-specific expression of the genes coding for the extracellular chitinases as well.
Fungal Resistant Plants Expressing Chimeric Chitinase Genes
Notwithstanding the many still unelucidated features concerning the nature and the role of hydrolytic enzymes in fungal resistance, some initial successes have been reported in providing plants with diminished sensitivity to fungal attack.
In U.S. Pat. No. 4,940,840, tobacco plants expressing a bacterial chitinase gene (i.e. the chiA gene from Serratia marcescens) have been shown to be less sensitive to the fungus Alternaria longipes.
In the International Patent Application WO 9007001 the plant species tobacco and canola, expressing a bean chitinase under regulation of a strong viral promoter or a plant promoter, appear to be less sensitive to two of the tested fungi, namely Botrytis cinerea and Rhizoctonia solani. It is not known, however, whether these plants are effectively resistant to other fungi as well.
In European Patent Application EP-A-0 292 435 it was suggested that resistance to certain classes of fungi may be conferred by the introduction of a gene that expresses chitinase in the plant tissues.
Mention was made of a preference in certain cases to target gene products into the mitochondria, the vacuoles, into the endoplasmic vesicles or other cell parts or even into the intercellular (apoplastic) spaces.
There was no teaching of the type of chitinase or of the preferred site of action of the chitinase, in order to obtain the desired effect.
EP-A-0 270 248 proposes a mechanism to target a bacterial gene (the .beta.-glucuronidase gene from E.coli) to the plant cell wall by using the leader sequence of the polygalacturonase gene from tomato. It was, inter alia, proposed to target chitinases or glucanases to the plant cell wall to combat fungal attack. Results were not shown, nor was indicated which hydrolytic enzymes should be used, or how intracellular plant proteins must be targeted outside the plant cell.
In the EP-A-0 332 104 genetic constructs are described comprising chemically regulative sequences derived from plant genes, among which the so-called PR-genes, including those coding for chitinase and glucanase. No results of fungal resistant plants were shown.